We previously identified unique peptide domains (designated LLP-1 and LLP- 2) in the cytoplasmic segment of the HIV-1 transmembrane (TM) envelope protein that have the structural and functional characteristics of natural cytolytic peptides and calmodulin (CaM)-binding proteins integral to cellular regulatory processes. Thus, we have proposed that the LLP segments of the HIV-1 TM protein can contribute to viral cytopathicity by interfering with CaM-dependent signal transduction and by perturbation of cell membranes and that the specific cytopathic effect observed will be dependent on the concentration and localization of the TM protein. A variety of biochemical and functional assays with LLP synthetic peptide homologs and analogs have confirmed the predicted CaM-binding/inhibitory and membrane perturbation properties and have also revealed rigid peptide structural requirements indicative a highly specific molecular interaction. We now propose to extend these LLP cytopathic studies to assays that more precisely monitor CaM functions and that are more relevant to actual HIV-1 infections. The specific aims of this proposal are; (i) to employ our panel of well characterized LLP synthetic peptide homologs and analogs to establish reliable cell culture models for monitoring alterations in signal transduction and in membrane permeability, (ii) to use the cellular assays developed in specific aim #1 to examine the role of the LLP domains of HIV- 1 TM protein expressed intracellularly as recombinant protein from plasmids containing complete native env gene or defined deletions or site-directed mutations in the TM gene segments, and (iii) to examine the cytopathic role of the LLP domains in the context of virus infected cells using specific infectious molecular clones of HIV-1 (native and defined TM mutations) and cell culture combinations optimized for monitoring single cell cytopathogenesis. Thus, these studies will employ state-of-the art technologies to test a novel concept in HIV-1 cytopathogenesis. The results of these studies will contribute to our overall understanding of HIV-1 cytopathogenesis. The results of these studies will contribute to our overall understanding of HIV-1 pathogenesis and may lead to improved strategies for management of HIV-1-related disease.